Prior work by our group has led to development of robust methods for production and purification of the anthrax toxin proteins: protective antigen, lethal factor, and edema factor. A large number of substitution and fusion proteins have been constructed to facilitate analysis and exploitation of these proteins. In the current year of 2015, we arranged through the NIH technology transfer office for many of these proteins to be distributed by Kerafast. Their website lists more than 10 of our protein variants that are available to researchers wishing to evaluate their utility. The anthrax toxin system is becoming recognized as a highly efficient tool for delivering proteins to the cytosol of eukaroytic cells, and the availability of the key proteins of this system should allow more researchers to take advantage of this tool. Recent years have seen increased effort directed toward development of protein-based anti-tumor drugs. Thus, a number of monoclonal antibodies have been approved by the FDA for the treatment of various cancers. A related and potentially more powerful approach which makes use of the high specificity binding of antibodies to tumor markers is the development of immunotoxins, fusions of tumor-specific antibodies to bacterial or plant toxins. In this laboratory, efforts have emphasized an alternative approach to achieving tumor cell specificity by using modified anthrax toxin. Anthrax toxin depends on proteolytic activation of the receptor-bound protective antigen protein (PA) by cell surface proteases. Replacing the furin-cleaved sequence with sequences recognized by matrix metalloproteases (MMP) or urokinase plasminogen activator has yielded tumor-specific anti-cancer fusion proteins that are efficacious in mouse tumor models. Both the wildtype and the modified PA assemble into oligomeric protein-conducting channels that delivers the anthrax toxin catalytic effector proteins lethal factor (LF) and edema factor (RF) to endosomes and then translocates them to the cytosol. For targeting of tumors, the native anthrax effector protein LF was replaced with a fusion containing the N-terminal PA-binding 254 amino acid-containing domain of anthrax toxin lethal factor (LFn) and the Pseudomonas aeruginosa exotoxin A (PE) catalytic domain (PEIII) to obtain the fusion protein FP59. The LFn domain delivers PEIII to the cytosol and PEIII transfers ADP-ribose to eukaryotic elongation factor 2 (eEF2), resulting in protein synthesis inhibition and cell death. This system is highly effective in achieving tumor-specific cell-surface PA activation and cytosolic delivery of PEIII. It has been successfully tested for a number of tumor types, and is expected to be active on nearly all types of solid tumors. In the reporting period of 2015, we worked to improve the specificity of the PA protein that is activated by MMPs. This PA-L1 protein is a key part of our tumor-targeting agents. Reference to the MEROPs database provided data on amino acid sequences that are optimal substrates for MMP2 and MMP9. Six of these sequences were inserted into PA, replacing the original furin cleavage site. The resulting proteins were purified and tested for susceptibility to various proteases including MMP2 and MMP9. While differences were observed, these were modest, and it was not evident that any of the new proteins were improvements over the original MMP-cleaved PA-L1 variants. The anthrax toxin system intracellular delivery system was used effectively in a study published in 2015 of Mycobacterium tuberculosis infections. The enzyme heme oxygenase-1 (HO-1) has been used as a marker to distinguish active from latent infections. The virulence-associated secreted protein ESAT6 was known to play a role in the induction of HO-1 in a murine macrophage cell line. It was shown in this study that infection of human macrophages with mutant M. tuberculosis strains lacking ESAT6 protein induced significantly lower HO-1 production. Delivery of ESAT6 recombinant protein into the cytosol of macrophages infected with ESAT6-deficient M. tuberculosis was accomplished using a fusion protein with the N-terminal fragment of the lethal factor of B. anthracis. This restored HO-1 induction and led to a decrease in MMP-1 secretion. This confirmed the key role of ESAT6 in the response to infection. Work during 2015 also facilitated the availability to the research community of the unique antibody S9.6, which recognizes DNA/RNA hybrids in a sequence-independent manner. This antibody and the producer hybridoma were licensed by NIH to Kerafast, which has distributed it very widely. The antibody has been adopted as a key reagent by researchers studying the normal and pathological roles of DNA/RNA hybrids. Transcriptional start sites typically have a region in which nascent mRNA is bound to one DNA strand, displacing the opposite DNA strand, producing what is termed an R-loop. Determining the location and abundance of R-loops provides important information regarding translational processes. Researchers have developed immunoprecipitation protocols for global analysis of R-loop sites. To facilitate further work with S9.6, we are developing methods for production of a single chain (scFv) version of the antibody. This protein will also be used by others to obtain crystal structures of the antibody bound to a DNA/RNA hybrid.